Down the membrane hole: Ion channels in protozoan parasites

Parasitic diseases caused by protozoans are highly prevalent around the world, disproportionally affecting developing countries, where coinfection with other microorganisms is common. Control and treatment of parasitic infections are constrained by the lack of specific and effective drugs, plus the rapid emergence of resistance. Ion channels are main drug targets for numerous diseases, but their potential against protozoan parasites is still untapped. Ion channels are membrane proteins expressed in all types of cells, allowing for the flow of ions between compartments, and regulating cellular functions such as membrane potential, excitability, volume, signaling, and death. Channels and transporters reside at the interface between parasites and their hosts, controlling nutrient uptake, viability, replication, and infectivity. To understand how ion channels control protozoan parasites fate and to evaluate their suitability for therapeutics, we must deepen our knowledge of their structure, function, and modulation. However, methodological approaches commonly used in mammalian cells have proven difficult to apply in protozoans. This review focuses on ion channels described in protozoan parasites of clinical relevance, mainly apicomplexans and trypanosomatids, highlighting proteins for which molecular and functional evidence has been correlated with their physiological functions.


Introduction
The establishment of lipidic boundaries in cells is a key evolutionary event that defines the existence of two distinct compartments, the intracellular and the extracellular space. The asymmetric distribution of molecules across the plasma membrane creates an electrochemical gradient that all living organisms use as energy source for the exchange of nutrients, water, and metabolites. Flow between compartments is mediated by ion channels and transporters, transmembrane proteins selectively carrying molecules across the lipid barrier. By controlling the flow of ions, channels regulate a diverse array of cellular functions including cell volume, motility, signaling, excitability, and cell death [1][2][3][4][5][6][7].
Phylogenetic analyses shows that ion channels are present in all prokaryotic and eukaryotic cells, with the common ancestors derived from bacterial porins and ABC transporters [8]. The type and abundance of channels and transporters often respond to the needs of each organism to cope with environmental conditions. Thus, the set of membrane proteins expressed by a cell equips them with the necessary tools to perform their function, for example, excitable cells expressed a higher number and diversity of ion channels compared with nonexcitable cells [9]. Structurally and functionally diverse, ion channels possess conserved core properties associated with the pore segments, and through their evolutionary history, they have acquired modular domains that regulate their activity and provide responsive elements linking the channels' gating with cell signaling events [10][11][12][13]. Traditionally, channels were classified based on their selective permeability to the most abundant ions in the intracellular and extracellular environments. As such, sodium, potassium, proton, chloride, and calcium channels cover most of the exchange between cells and their surroundings. Channels can be also classified by their gating or activation mode, depending on their response to changes in voltage, binding of intracellular or extracellular ligands, or interaction with the membrane lipids. In the past 20 years, the increasing knowledge on the role of channels in sensing modalities gave origin to a new classification based on their ability to detect oxygen, acid, mechanical stimuli, temperature, etc. [14]. These types of channels are particularly interesting in the context of host-parasite interactions, as they could be involved in the detection of environmental conditions that regulate developmental transitions.
The first crystallographic structure of a potassium channel was obtained in 1998 when the small bacterial KcsA channel was described [15]. Since then, hundreds of channels have been characterized in bacteria, fungi, plants, and mammals [14,16,17], and their roles in the most diverse physiological functions from survival to death have been established. In contrast, only a few channels have been identified or even partially characterized in protozoan parasites (Tables 1 and 2). Despite the relevance of ionic control in protozoans, our knowledge of the specific molecules responsible for the permeability of ions across the plasma membrane and the intracellular organelles is still limited and fragmentary. Low sequence homology with known channels, lack of response to selective blockers, and technical difficulties of direct electrophysiological recordings in small motile cells are some of the challenges that limit our knowledge of ion channel physiology in these parasites [18]. In the following sections, we will summarize current information describing the physiological role of ion channels as regulators of cellular functions and host-parasite interaction. We will describe channels residing in the plasma membrane and main intracellular organelles, since channel localization and abundance is a determinant of ion exchange between compartments. We will dedicate a special section to proteins produced by the parasites and exported to the membranes of host cells where they mediate nutrient exchange. Finally, we will propose future research directions to increase our understanding of ion physiology in unicellular parasites while offering insights on their potential as selective drug targets.

Physiological roles of ion channels in protozoans
The ionic composition of the cytosol and organelles in protozoan parasites is similar to that of mammalian cells, with K + as the predominant intracellular cation, Ca 2+ concentrations around 100 nM and variable amounts of Cl − and organic anions [19]. Consequently, we must assume the presence of ion channels and transporters sustaining the electrochemical gradients and the membrane potential. Genome-wide and phenotypic analyses conducted in Plasmodium showed a reduced number of channels and transporters expressed in the parasites compared with bacteria and mammalian cells [20][21][22], suggesting the selection of a minimum transportome that supports the survival and replication of protozoans adapted to a parasitic lifestyle [23][24][25][26]. The lack of redundancy on the transport mechanisms also suggests the essentiality of these proteins, but conclusive functional evidence is needed to demonstrate this hypothesis.
The Na + /K + ATPase is the predominant pump operating in the plasma membrane of mammalian cells [27,28], but its identification in unicellular parasites has been elusive, with some reports of its function in Leishmania [29]. Like plants, protozoans abundantly express H + - TgVDAC TGME49_263300 Toxoplasma gondii Mitochondria [195] ATPases and regulate most of their membrane processes based on H + motive force instead of Na + or K + fueled transport. Therefore, proton-dependent membrane potential and pH regulation are strictly linked in the parasites, while other monovalent cations are required to support the activity of pumps and transporters that control nutrient exchange (Fig 1). The membrane potential (Vm) of multiple protozoans has been measured and is generally more negative than in nonexcitable mammalian cells, with values averaging −80 to −120 mV [30][31][32][33][34][35]. Multiple studies have confirmed the H + dependency of the membrane potential and a relative insensitivity to the presence of monovalent cations in the media, with some exceptions. T. brucei bloodstream trypomastigotes and P. falciparum trophozoites are strongly depolarized in high K + media [30,35] and hyperpolarized in the presence of Ba 2+ , suggesting the presence of a channel mediating the influx of K + to counterbalance the activity of the membrane H + -ATPases. Chloride conductance also influences membrane potential, and it has been postulated that an anion channel may be responsible for the Cl − uptake and concomitant depolarization observed in Leishmania [32] and Plasmodium [36]. The hyperpolarized Vm observed in protozoan parasites compared with mammalian cells may provide buffering capacity for H + mobilization, necessary to maintain tight control of the intracellular pH, as it has been shown in bacteria [16,37]. Remarkably, protozoans are able to keep their intracellular pH constant under challenging external conditions such as the incorporation into phagolysosomes [38,39] or the transit through the intestinal track of insect vectors [40]. Furthermore, cues such as media acidification are used as signals to trigger their differentiation [41,42] and to modify In nonexcitable mammalian cells, the resting membrane potential (Vm) is maintained at values close to −30 mV by the coordinated activity of ion channels and transporters. This membrane potential is driven by a high permeability to K + caused by the abundant expression of leak K + channels. Na + influx via channels, exchangers, and cotransporters is counterbalanced by the activity of the Na + /K + ATPase, main membrane pump found in vertebrates. Cl − , the most abundant intracellular anion, is regulated by electroneutral transporters such as the Cl − -Na + -K + cotransporter and by ClC channels. While Ca 2+ influx via VGCCs does not cause a significant change in the membrane potential directly, the cascade of signaling events associated with Ca 2+ entry affects expression and trafficking of the channels and transporters mentioned above. In trypanosomatids, and other protozoan parasites studied to date, membrane potential is driven by a proton motive force generated by the activity of H + ATPases, establishing a value of about −100 mV for T. cruzi epimastigotes. Depending on the life stages analyzed, K + plays a secondary role on Vm maintenance and the hyperpolarization supports the influx of K + by channels and/or via exchangers. No Na + channels have been identified but permeation occurs through NSCCs and exchangers, while efflux is mediated by ENa ATPases. Figure created with [46,47]. This is, perhaps, the most relevant signaling event in parasite's physiology since Ca 2+ oscillations control gene expression, differentiation, motility, invasion, and egress from host cells [4,[48][49][50][51][52][53][54][55]. Calcium signaling also determines the fate of cells, stimulating replication and triggering cell death [56][57][58][59][60][61]. While essential cellular functions are strongly dependent and tightly controlled by ionic conditions, our understanding of the molecules that regulate ion exchange in the parasites is limited. In the following sections, we will summarize the current knowledge of ion channels in protozoans focusing on proteins that have been molecularly identified and functionally analyzed. For clarity, we will use the localization of the channels as their primary classification criteria, with some exceptions due to the complexity of the topic.

Plasma membrane channels
The separation of the intracellular and extracellular environments by a selectively permeable lipid barrier implies the need for transmembrane proteins facilitating the exchange of ions and other molecules. Na + and K + gradients, together with Ca 2+ oscillations, are major stimuli that regulate cell volume, cell survival, and signaling [2]. The predominance of Na + in blood and tissue fluids and the intracellular acidification observed in the absence of extracellular Na + [62-64] suggest the presence of Na + channels in the plasma membrane of extracellular parasites, but their molecular identity is still obscure. Curiously, ouabain-sensitive Na + /K + ATPases, extremely abundant in mammalian cells, do not seem to play a significant role in ion homeostasis in protozoans; instead, Na + pumps are ubiquitously expressed. In Plasmodium falciparum PfATP4, initially described as a Ca 2+ pump [65], was later demonstrated to be a plasma membrane Na + efflux pump [66,67] associated with resistance to antimalarials [68,69]. TgATP4, a homolog found in Toxoplasma gondii, controls the cytosolic concentration of Na + [70]. In Trypanosoma cruzi, a Na + ATPase insensitive to ouabain (TcENA) is more abundantly expressed in epimastigotes and trypomastigotes compared with intracellular amastigotes [71,72]. Trypanosoma brucei and Leishmania [73] also possess P-type Na + ATPases similar to TcENA [74,75]. The primary role of ENA ATPases is to maintain a Na + gradient that fuels secondary transport of nutrients, and their function is necessary linked to the influx of Na + via ion channels. Despite the indirect evidence of their presence, not a single Na + selective channel has been identified in protozoan parasites. It is possible that nonselective cation channels such as TcCat [76] serve as permeation pathways for both, Na + and K + , depending on the electrochemical gradients, thus fulfilling the role of Na + channels, but the complete absence of this type of channels is unlikely and further research exploring this question is needed.
While in mammalian cells K + permeability is a major contributor to membrane potential maintenance [77], this ion plays a secondary role in protozoans, with proton-motive forces driving most of the resting membrane potential [30,31,[33][34][35]78]. K + , as the most abundant cytosolic cation, is essential for the function of H + /K + exchangers, and its concentration has a profound effect on the regulation of cell volume, enzymatic activity, and cell death [1,77]. Analysis of protozoan genomes predicts the presence of sequences encoding for voltage and calcium-gated potassium channels as well as inward rectifiers [79], and a discrete number of channels have been cloned and characterized (Tables 1 and 2). Electrophysiological recordings of proteoliposomes containing membranes isolated from T. cruzi epimastigotes showed the presence of at least two distinct potassium pathways: a larger channel with a conductance of around 100 pS, mostly permeable to K + , and a nonselective cation channel, with lower conductance and slight rectification [80]. This channel shares some functional characteristics with TcCat, a nonselective cation channel expressed in all life stages of T. cruzi [76], including blockage by divalent cations and similar conductance, but they differ in their selectivity and permeability sequence. These functional differences may indicate the presence of two different nonselective cation channels or could correspond to the same channel whose activity is modulated by the lipidic environment and or/signaling, often lost when the proteins are expressed in heterologous systems. The nonselective nature of TcCat agrees with the absence of a conserved selectivity filter, a feature found in all K + selective channels [15,81]. The channel localizes to the plasma membrane of the parasites and is required for volume regulation under hyperosmotic conditions [76], a function often dependent on the mobilization of K + . A second K + channel was described in T. cruzi and showed features of a calcium-activated K + channel (TcCAKC). This protein plays a central role in regulating intracellular pH and membrane potential in T. cruzi [82] and its ablation in epimastigotes induced hyperpolarization and cytosolic alkalinization, as well as an increase in the rate of proton extrusion. Importantly, TcCAKC knockout parasites were unable to differentiate into trypomastigotes, severely impairing their infectivity and emphasizing the role of K + homeostasis in parasite fitness. Previously, it has been shown that trypomastigotes membrane potential is more sensitive to extracellular K + than that of epimastigotes [31], and this is also the case for T. brucei [30]. TbK1 and TbK2 form heterodimeric K + channels required for membrane potential regulation and cell survival in T. brucei bloodstream forms, while being dispensable in procyclic forms. These results suggest a tighter control of K + conductance at the plasma membrane in the bloodstream stages [83] in vitro but raise the question of whether this ion could have a more drastic influence on parasites survival when evaluated in vivo. Indirect evidences suggest the presence of calcium-activated K + channels [84] and nonselective cation channels in Leishmania amastigotes and promastigotes [85,86], but no molecular or biophysical characterization of these putative proteins has been reported.
In apicomplexans, a limited number of plasma membrane channels have been characterized (Table 2). PfKch1 is a six-transmembrane domain protein with a conserved selectivity filter sequence that is expressed throughout the erythrocytic cycle, with higher expression in trophozoites and schizonts [87]. Interestingly, as the parasites mature, the protein could be detected in the plasma membrane of infected red blood cells (RBCs), suggesting the export of the channel during later developmental stages. PfKch2 is a smaller protein, found in the plasma membrane of schizonts and merozoites [87]. Attempts to knock out these channels have failed and exposure to calcium-activated K + channel blockers like clotrimazole had a potent antimalarial effect, suggesting the essentiality of PfKch1 and PfKch2. Recently, expression of these two proteins in Saccharomyces cerevisiae showed their membrane localization and ability to form functional channels when reconstituted in lipid bilayers [88]. Rb + uptake experiments in Plasmodium berghei validate the role of the homolog PbKch1 and PbKch2 as K + permeation pathways. While PbKch2 knockout did not have a drastic effect in asexual or sexual developmental stages [89], PbKch1-null parasites, while still able to develop asexual stages, failed to produce oocysts, resulting in a 98% reduction of transmission through mosquitoes [90]. This observation suggests a potential therapeutic target that could interrupt transmission and argues for a complete in vivo characterization of K + channels in Plasmodium that could offer new insights into the role of ion regulation in the parasite's life cycle.
Changes is free cytosolic Ca 2+ also facilitate cell motility, differentiation, and tumorigenesis [95][96][97]. In intracellular parasites, host cell invasion [4,48,98] and egress are also Ca 2+ dependent [54,99-103]. The evidence of Ca 2+ uptake from the extracellular medium and the interaction of this ion influx with the intracellular stores is overwhelming, pointing out the presence of Ca 2+ -permeable channels at the plasma membrane [4,53,104-108]. L-type nifedipine-sensitive Ca 2+ conductances have been described in T. cruzi [109][110][111], Leishmania [105,110,112], and T. gondii [4]. In agreement with this functional evidence, putative genes for L-type voltage-gated Ca 2+ channels (VGCCs) have been identified in the genome of these parasites [113]. Rodriguez-Duran and colleagues recorded the activity of plasma membrane vesicles purified from T. cruzi epimastigotes and reconstituted into liposomes [110]. They established the presence of Ca 2+ currents activated by sphingosine, miltefosine, and Bay K 8644, an activator of Ltype VGCC. While all the sphingosine-stimulated activity was blocked by nifedipine, this compound only partially blocked currents elicited by miltefosine, suggesting either a difference in the activation mechanism or, more plausibly, the presence of more than one type of channel. While the permeability sequence agrees with previously characterized L-type VGCC, the channels in T. cruzi are not activated by voltage, indicating a difference in the gating mechanism. The only sequence identified in the T. cruzi genome as a putative VGCC is TcCLB.504105.130, with homologs in Leishmania and T. brucei (Table 1). Ca 2+ uptake stimulated by sphingosine and miltefosine has also been described in L. mexicana and L. donovani [105,112]. In T. brucei, a putative VGCC called FS-179 has been localized to the flagellar membrane, but no functional studies to verify the biophysical properties of this protein have been conducted [59,114]. Definitive molecular evidence linking the nature of these currents with specific proteins is an important step to understand how trypanosomatids regulate Ca 2+ flux at the plasma membrane.
Plasmodium Ca 2+ currents are refractory to L-type channel blockers [107,115], and no putative VGCC sequences were found in its genome. Instead, possible homologs to transient receptor potential (TRP) channels are present in P. falciparum, P. vivax, and P. knowlesi [113], but their expression and function remain uncharacterized. In T. gondii, a recently described TRP channel, TgTPPL-2, mediates Ca 2+ influx in tachyzoites [52]. This nonselective cation channel, localized in the plasma membrane and the endoplasmic reticulum (ER), contributes with approximately 50% of the uptake stimulated by physiological extracellular concentrations of Ca 2+ and is not blocked by nifedipine, indicating that additional channels must be responsible for the residual Ca 2+ influx [52]. Parasites in which TgTPPL-2 expression has been downregulated showed reduced growth and impaired invasion and egress. When expressed in HEK-3KO cells, this channel also mediated currents at the ER, suggesting its possible role in the release of Ca 2+ from intracellular stores [52]. This work provided the first molecular evidence linking a membrane channel with the observed Ca 2+ influx in T. gondii, but it also underscores the need for electrophysiological recording methods that can be applied directly on the parasites, as the biophysical properties observed in heterologous expression system may not fully represent the activity of the native channels.
Chloride is the most abundant anion in nature, with extracellular concentrations of 120 mM and intracellular values ranging from 10 to 40 mM, depending on the cell type [6]. Plasma membrane chloride channels are responsible for the permeation of Cl − and other anions, counterbalancing the net positive charges resulting from cation flows, and ensuring electroneutrality of cotransporters. As such, chloride channels are key regulators of cell volume, excitability, contractility, cell survival, and transepithelial transport [3,116,117]. Recently, the role of Cl − as a regulator of cell signaling and cell proliferation has been associated with the progression of cancer and immune activation [3,118]. Chloride channels can be activated by voltage (CLC channels), volume (volume-regulated anion channels (VRACs)), ligands (GABA and glycine), or Ca 2+ (calcium-activated chloride channels (CaCCs)). A particular type is the CFTR (cystic fibrosis transmembrane conductance regulator), epithelial chloride channel regulating secretion and water balance in multiple organs, and whose loss of function mutations underlie cystic fibrosis pathologies [119,120]. Localized either at the plasma membrane or in intracellular organelles, their function is required for adequate ion homeostasis.
In protozoans, there is limited information regarding expression and activity of plasma membrane Cl − channels, with only one protein characterized in Entamoeba [121] ( Table 2). EhCLC A is localized in the plasma membrane of trophozoites and shares homology with CLC-2 channels, including its permeability sequence and lack of modulation by pH, supporting its role as an electrogenic Cl − channel and not as an antiporter. A second sequence named EhCLC B was also identified, but its characterization is still pending [121]. Patch clamp recordings of Giardia intestinalis trophozoites showed the activation of chloride currents compatible with CLC-2-type channels [122]. pH had no effect on the magnitude of the currents but did modify the voltage sensitivity. Unlike EhCLC A, only responsive to SITS (4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid), the channels in G. intestinalis were blocked by canonical Cl − channel blockers DIDS (4,4 0 -diisothiocyanatostilbene-2,2 0 -disulfonic acid) and niflumic acid but not by SITS. The putative genes encoding for these channels have not been identified, but G. intestinalis mRNA injection in Xenopus laevis oocytes produced at least three types of chloride currents with different profiles: a CLC-2-type, a Ca 2+ -activated, and a volume-responsive chloride channel. These three channels showed distinct voltage dependency, gating, and selectivity, providing solid functional evidence of their independent nature. The difference in their pharmacological profiles is a useful tool for functional analysis since the molecular identity and localization of these channels have not been established yet. Injection of mRNA from Leishmania amazonensis also elicited anionic currents sensitive to DIDS and niflumic acid, but the interpretation of this data is difficult due to the experimental conditions and the presence of endogenous Clcurrents in the oocytes [123]. Certainly, several classes of Cl − channels must be present in the plasma membrane of protozoan parasites. Their molecular identification and characterization would provide a missing piece in the proposed model of membrane exchange that supports ionic balance and secondary transporters required for survival, homeostasis, and infectivity [45,124].

Aquaporins
Movement of water across membranes is a fundamental cellular process necessary for cell survival. Net flow of water results from changes in ion and osmolyte concentrations between the intracellular and the extracellular space, which causes an osmotic imbalance and drives fluid movement. Most of the water mobilization occurs through specialized water channels called aquaporins (AQP). Initially found in human erythrocytes and kidney cells [125], they have been described in most organisms, and their main role is to regulate cell volume [1,125,126]. Water channels can be classified in three groups: canonical aquaporins, only permeable to water; aquaglyceroporins that allow flow of glycerol, urea, and other small molecules; and superaquaporins, found intracellularly mostly in mammals [127].
In protozoan parasites, the number, expression, and localization of AQP varies from only one in T. gondii [128] and P. falciparum [129], to five putative AQPs in Leishmania [130][131][132][133]. It has been proposed that this variation correlates with the environmental challenges faced during the life cycle and the capacity of the parasites to respond to them [134]. In T. brucei, TbAQP1 and TbAQP2 localize to the flagellar membrane and flagellar pocket, respectively (Table 1), while TbAQP3 is restricted to the plasma membrane. In L. major, LmAQP1 is also found in the flagellum, suggesting a role of this structure in sensing osmotic conditions. In T. cruzi, the only characterized AQP, TcAQP1, mediates water flow to the contractile vacuole complex (CVC), an organelle that regulates cell volume in the parasites (Fig 2) [135,136]. Functional studies showed that most AQPs found in protozoan parasites, except for TcAQP1 [135], are permeable to water as well as glycerol and other small solutes including urea, ammonia, and arsenite. Glycerol permeation through AQPs could play a role in regulating glycosomal activity and energy metabolism [137,138].
AQPs are considered major intrinsic proteins (MIPs), membrane channels that mediate the exchange of water and small solutes. As such, they also play a role in permeability to antiparasitic compounds and have been postulated as potential drug targets [130,139]. In Leishmania [132] and T. brucei [140], AQPs mediate the uptake of trivalent antimonial compounds and down-regulation of LmAQP1 correlates with resistance to antileishmanial drugs such as Pentostam [132,141], while TbAQP2 expression is linked to melarsoprol-pentamidine cross-resistance [142].
Evidently, the physiological functions of AQPs in protozoans go well beyond cell volume regulation, possibly also regulating metabolisms and drug susceptibility as well. Some protozoans can manipulate the expression of AQPs in the host cells. Hepatocytes infected with P. vivax showed increased AQP3 expression, and active recruitment of the host's AQP3 to the In T. cruzi, cell volume regulation is controlled by the CVC and the ACCS (reviewed in [247]). Both organelles are rich in ions accumulated by the activity of pumps located on their membranes. Upon hypoosmotic stress, ACCS fuse with the CVC transferring Pi, Ca 2+ , and AA. This concentration of osmolytes drives water into the vacuole via AQP1, causing swelling of the bladder that can be sensed by a mechanosensitive channel (MscS). Contact between the CVC membrane and the flagellar pocket induces the formation of a transient pore that mediates the discharge of water and osmolytes to the EC space. Some ions, like Pi, are not released but instead recovered by transporters (Pho1). These events are accompanied by increase in cAMP and a rise in cytosolic Ca 2+ required for volume recovery. Trypanosoma brucei does not possess a contractile vacuole and regulates cellular volume via aquaporins, with AQP2 residing in the flagellar membrane and AQP3 at the plasma membrane.

PLOS PATHOGENS
parasitophorous vacuole membrane (PVM) during liver and blood stage development [143,144]. Down-regulation of AQP3 by RNAi or inhibition by auphen drastically reduced the parasite load in liver cells. Analysis of P. falciparum-infected RBCs showed similar recruitment of AQP3 to the PVM, and treatment with auphen reduced parasitemia by 75% [144]. While the mechanism of translocation from the plasma membrane to the PVM has not been established, the temporal profile of recruitment early during schizont and erythrocytic stages suggest a role of the channel as a nutrient uptake pathway [143]. Recently, transcriptomic analysis of permissive and nonpermissive hepatocytes has identified AQP9 as a host cell factor required for sporozoite invasion [145]. Silencing or chemical inhibition of host APQ9 drastically reduced hepatocyte invasion by P. falciparum and P. berghei without compromising further intracellular development of liver stages. The role of AQP9 during invasion is unclear, but it is possible that osmotic changes at the plasma membrane, elicited by water influx, could indirectly modify actin polymerization observed during invasion [145] an interesting hypothesis that requires further exploration.

Mitochondrial channels
In mammalian cells, mitochondrial morphology, distribution, and functions have been extensively studied [146,147]. Originated by an endosymbiotic event [148], mitochondria possess a double-membrane structure that surrounds the matrix, where most of the proteins and the mitochondrial DNA (mtDNA) reside. The outer mitochondrial membrane (OMM) separates the cytosol from the intermembrane space, regulating interactions with other organelles and the exchange of metabolites and proteins [149]. The inner mitochondrial membrane (IMM) harbors metabolic pathways involved in oxidative phosphorylation, fatty acid oxidation, and small molecule transport [146]. InvaginationsAU : PleasecheckandconfirmthattheeditstothesentenceInva of the IMM give origin to the cristae, structures that fold toward the matrix and whose morphology and abundance depend on the presence of multimeric complexes including the ATP synthase and other electron transport chain (ETC) components, the mitochondrial contact site and cristae organizing system (MICOS) [150,151], and the protein importing systems [152].
Often referred to as the powerhouse of the cells for their role in ATP synthesis, mitochondrial functions expand beyond that of oxidation of fatty acids, redox balance, regulation of cell death, and calcium signaling modulation. The number, shape, and localization of these organelles, collectively known as mitochondrial dynamics [146,147], respond to cellular signals such as changes in metabolic demands, immune challenges, and the presence of intracellular pathogens, among others [153][154][155].
In protozoans, mitochondrial morphology, function, and dynamics are dependent on the developmental stages of the parasites. Trypanosomatids possess a single mitochondrion, with its mtDNA organized in maxi and mini circles forming the kinetoplast. The OMM and IMM extend throughout the body of the parasites, and the abundance of cristae varies depending on life stages. T. brucei bloodstream trypomastigotes are mostly glycolytic, and their mitochondria, although essential [156,157], are not as developed as in the procyclic forms that rely heavily on oxidative metabolism [158]. T. gondii tachyzoites [159,160] and Plasmodium blood and liver stages also have a single mitochondrion that undergoes extensive branching and remodeling during cell division [161,162]. In contrast, Cryptosporidium, Entamoeba, and Giardia possess mitochondria that have lost most of their genome and are unable to perform oxidative phosphorylation due to an incomplete ETC [163-167]. These organelles, called mitosomes, are required for parasite survival and play essential roles in energy production by substrate-level phosphorylation, lipid metabolism, and assembly of Fe-S clusters. Similarly, in T. vaginalis and other trichomonads, organelles known as hydrogenosomes [168] evolved from a mitochondrial-like ancestor but completely lost their genome and most ETC-related proteins [167,169]. Instead, they produce ATP by substrate-level phosphorylation and protons by the activity of an Fe hydrogenase [170]. Hydrogenosomes possess a robust protein import machinery with similar components to those found in mammalian mitochondria [171][172][173].
Despite the vast diversity of forms and functions, mitochondria and related organelles in protozoans possess a membrane potential that must be maintained for the parasites to survive. In T. brucei, the mitochondrial membrane potential ( mt Vm) has been estimated to be approximately . Under physiological conditions VDAC is a nonselective porin, preferentially permeable to chloride but also able to conduct ATP, K + , and Ca 2+ depending on the transmembrane voltage [186]. The channel allows for the passage of noncharged solutes from 3 to 6 kDa in size [187], constituting an important regulator of metabolic flux between the cytosol and the mitochondria [188]. The role of VDAC has been extensively studied in eukaryotes and includes the regulation of mitochondrial metabolism, apoptosis, protein import, and modulation of mitochondrial morphology [189][190][191], but the extent to which these discoveries apply to parasitic protozoa is unclear. Importantly, VDAC plays a fundamental role in the interaction of mitochondria with other organelles. VDAC1 and VDAC2 interact with IP 3 receptors localized at the mitochondria-ER contact sites (MERCS) and is required for Ca 2+ flux [192,193], while VDAC2 and ryanodine receptor (RyR) 2 regulate Ca 2+ dynamic in cardiac cells [194]. Recently, Mallo and colleagues showed that in T. gondii, TgVDAC localizes to MERCS, and depletion of the channel reduces the number of contact sites, leading to ER fragmentation [195]. Down-regulation of TgVDAC in tachyzoites caused replicative defects accompanied by aberrant mitochondrial morphology. Surprisingly, while TgVDAC depletion disrupted protein import to the mitochondria, no defects in Ca 2+ homeostasis were observed and only a marginal decrease in ATP/ADP ratio was reported [195]. These results support a partially conserved role of TgVDAC in T. gondii, mainly regulating mitochondrial dynamics and inter-organelle contact.
In T. brucei, two putative VDAC sequences have been identified (Table 1) [196]. The porin was found as oligomers of an apparent molecular weight of 212 kDa, corresponding to a hexameric or heptameric complex as previously reported in other cells [189]. Knockout of VDAC1 in procyclic forms leads to growth defects only under conditions that favor oxidative phosphorylation, showing no difference in fitness if glucose is present. This study suggests that VDAC is not required for survival in parasite stages that rely in glycolytic production of ATP [197]. In agreement with metabolic differences previously reported [198,199], Singha and colleagues showed a 5-fold higher expression of VDAC in procyclics compared to bloodstream forms [200], but in this work, authors reported a decrease in growth and mitochondrial ATP production in both life stages upon down-regulation of VDAC [200]. Despite these discrepancies, it is clear that VDAC plays a role in metabolite import to the mitochondria in T. brucei.
Putative VDAC sequences are present in the genome of T. cruzi, Leishmania, and Plasmodium awaiting for experimental interrogation of their roles in the biology of these parasites.
The proton motive force across the IMM responsible for the membrane potential also creates a driving force for Ca 2+ uptake, making mitochondria one of the most important Ca 2+ stores in the cell. Through fine-tuning of signaling and Ca 2+ modulation, the mitochondria control cell fate, triggering either necrosis or apoptosis in response to level and duration of cytosolic Ca 2+ increases. The accumulation of Ca 2+ in the mitochondria requires the permeation through the OMM, possibly mediated by VDAC [192][193][194], and the IMM, via the mitochondrial calcium uniporter (MCU) [201]. The MCU is a hetero-oligomeric protein complex whose constituents reside embedded in the IMM and protrude into the intermembrane space. The MCU is the pore-forming protein that mediates Ca 2+ import. It associates with regulatory proteins MICU1 and MICU2, as well as EMRE (essential MCU regulator), an essential subunit that facilitates the interaction of MCU and MICUs (reviewed in [202]). An alternative poreforming subunit, MCUb, can associate with MCU, reducing its transport capacity, as MCUb is not able to permeate Ca 2+ in mammalian cells. In trypanosomatids, mitochondrial Ca 2+ uptake mediated by an MCU-like mechanism was postulated in 1989 in T. cruzi epimastigotes [203,204] and later also found in Leishmania [175] and T. brucei [174]. Molecular identification of the proteins responsible for this Ca 2+ influx will follow 30 years later, with the characterization of the MCU complex in T. brucei [205,206] and T. cruzi [207][208][209]. In these parasites, the core subunits MCU, MCUb, and MICU1/2 have been localized to the mitochondria, where they were shown to be essential for Ca 2+ uptake and bioenergetic functions. No homologs of EMRE have been found in trypanosomes. Surprisingly, while mammalian MCUb is a negative regulator of Ca 2+ uptake, T. cruzi homolog TcMCUb facilitates mitochondrial Ca 2+ uptake, and its ablation negatively impacts parasite survival, differentiation, and infectivity [207]. T. brucei homolog (TbMCUb) has similar functions, and its down-regulation impairs mitochondrial Ca 2+ uptake without causing changes in mt Vm or cell growth [206]. Two additional subunits TbMCUc and TbMCUd, uniquely identified in T. brucei, are expressed and localized to the mitochondria, directly interacting with TbMCUb and TbMCU [206]. RNAi of any of the TbMCU subunits caused a decrease in Ca 2+ uptake without further detriment to the cells, unless they were deprived of glucose. While this may suggest a certain degree of redundancy on the formation of the MCU complexes in T. brucei, the inability to complement the function with alternative subunits indicates unique roles for each of the components. Supporting this idea, the elimination of TcMCU or TcMCUb in T. cruzi impacted the ability of the mitochondria to capture Ca 2+ without affecting the membrane potential, but only TcMCUb knockouts showed reduced growth, respiration rates, differentiation, and infectivity [207]. A similar phenotype was observed when MICU1 or MICU2 were eliminated, but their role in the regulation of the MCU differs from reports in mammalian cells, as T. cruzi MICUs seem to work as stabilizers of the complex rather than as gatekeepers [210].
The activation of the MCU causes a transient Ca 2+ increase in the mitochondria in the order of 50 to 100 mM, while the average cytosolic concentration is around 100 nM. This apparent discrepancy, together with the reported low affinity of the MCU [201], posed the question of how was the Ca 2+ uptake possible. Multiple evidences have shown that the influx occurs at cellular microdomains where the mitochondria can interact with the ER [211] or with the plasma membrane [212] and that the activation of IP 3 R, RyR or other channels provides a local Ca 2+ increase that can be captured by the MCU complex [192,193,213]. In trypanosomatids, IP 3 Rs have been found in the acidocalcisomes instead of the ER [214,215], suggesting a possible interaction between these Ca 2+ -rich organelles and the mitochondria [216,217].
The trypanosome MCU complex fulfills crucial roles in Ca 2+ uptake and metabolism, as it has been shown in mammalian cells, but it also seems to affect replication, differentiation, and infectivity of the parasites. The presence of new MCU subunits, found exclusively in trypanosomes, may offer a window for selective targeting of the parasites, as Ca 2+ homeostasis has been postulated as a potential area for drug development [218].
In apicomplexans, motility, invasion, and egress are strongly dependent on Ca 2+ [4,55,106,219]. Despite some reports of sensitivity to the MCU blocker ruthenium red [220], there is no genomic evidence of an MCU complex in either Toxoplasma or Plasmodium, suggesting the loss of these proteins, as it has been also observed in mitosome-harboring protozoans and some yeast [221]. Whether mitochondrial Ca 2+ plays an important role in regulating metabolic and cellular functions and how is this ion pool managed in apicomplexans are still pending questions.

Channels in other organelles
Intracellular compartments maintain their composition by harboring a differential set of proteins that support specialized functions. In protozoans, ER, acidocalcisomes, and mitochondria are the main intracellular Ca 2+ stores, controlling the amount of free cytosolic Ca 2+ and cellular functions linked to Ca 2+ -dependent signaling [216]. In apicomplexans, two additional organelles participate in Ca 2+ homeostasis: the food vacuole of Plasmodium [222] and the plant-like vacuole in T. gondii [223], while in T. cruzi, the CVC has been postulated as a Ca 2+ store [224]. Acidocalcisomes are acidic organelles rich in phosphate found in trypanosomatids [98, 225,226], apicomplexans [227][228][229], and other microorganisms [230] including bacteria [231], nonparasitic protozoa [232], and algae [233]. In T. brucei and T. cruzi, they accumulate calcium through a Ca 2+ -ATPase abundantly expressed in their membrane [234][235][236], while efflux is mediated by IP 3 receptors [214,215]. Nuclear patch clamp recordings of DT40-3KO cells expressing TbIP 3 R (T. brucei IP 3 receptor) showed that the channel is modulated by phosphate, with an increase in the current and open probability in the presence of orthophosphate or pyrophosphate, while polyP3 and acidic pH had an inhibitory effect [237]. These results suggest that TbIP 3 R could be activated under hypoosmotic stress conditions, when the acidocalcisomes show alkalinization and polyP hydrolysis [238,239]. Importantly, down-regulation of TbIP 3 R reduces cell growth in procyclic and bloodstream forms, also decreasing infectivity in vivo [215]. Similarly, knockout of the IP 3 R in T. cruzi causes severe reduction of epimastigotes growth and intracellular amastigote replication, decreasing metacyclogenesis and infectivity. The elimination of TcIP3R has a profound effect in mitochondrial functions with decreased Ca 2+ uptake, oxygen consumption and citrate synthase activity, increased AMP/ ATP ratio, and induction of autophagy. This phenotype is remarkably similar to the one observed in parasites where the MCU subunits were ablated [207,209], strongly supporting the hypothesis of acidocalcisome-mitochondrion contacts regulating Ca 2+ dynamics [240] and providing additional evidence of the role of Ca 2+ channels as regulators of parasite fitness in trypanosomes [216].
In apicomplexans, despite the responses elicited by IP 3 [49,241] and cyclic ADP ribose [242], and the presence of PLC signaling pathways [243,244], no molecular evidence of IP 3 R or RyR expression has been found, suggesting that the release of Ca 2+ from intracellular stores could be mediated by a highly divergent type of IP 3 R or by a different type of IP 3 -responsive channel [245], exciting hypotheses that require further experimental support.
Acidocalcisomes accumulate large amounts of phosphate as polyP polymers. These anionic molecules interact with inorganic and organic cations, translocated to the lumen by cation transporters and exchangers (reviewed in [240]). An inwardly rectifying K + channel (TbIRK; Table 1) has been localized to the acidocalcisomes of T. brucei, contributing to the cation influx [246]. As expected by the lack of a conserved selectivity filter, this channel has a relative permeability P K /P Na around 7, quite small considering that selective K + channels have a preference for K + over Na + of hundred to thousand folds [15]. Its down-regulation had no effect on the growth of procyclics but could not be evaluated in bloodstream forms due to low RNAi efficiency, leaving an open question about the physiological role of this channel.

PLOS PATHOGENS
In T. cruzi, acidocalcisomes have been shown to fuse with the CVC, transferring ions and proteins to this compartment [136]. The accumulation of ions and organic osmolytes in the CVC attracts water, causing periodic discharge of its content to the extracellular space and regulating the parasite's volume (Fig 2). The CVC operates in cycles of systole and diastole, which, under isotonic conditions, cause a discharge event approximately every 90 seconds. The frequency of CVC emptying depends on the osmotic conditions, increasing under hypoosmotic stress [247]. It has been postulated that sensing of the filling state must be mediated by mechanosensitive elements. Recently, a K + -and Ca 2+ -permeable mechanosensitive channel residing in the CVC (TcMscS) has been identified in T. cruzi. The channel participates in osmotic stress responses, Ca 2+ homeostasis and regulation of cell differentiation and replication [111]. A homolog in T. brucei (TbrMscS) shows similar activity but is localized to the mitochondria. Putative mechanosensitive channels have been found in the genome of Apicomplexans, Trichomonads, and Amebae [248], but no functional studies have been conducted so far. Recently, a two-pore channel (TgTPC) has been identified in the T. gondii [57]. TPCs are cation channels abundantly expressed in the endolysosomal system [249] where, due to their mechanosensitivity, they participate in organelle biogenesis, osmotic responses and volume regulation [250,251]. In T. gondii, TgTPC is exclusively found in the apicoplast, an endosymbiotic organelle that harbors multiple metabolic pathways required for parasite division [252][253][254]. Li and colleagues showed that TgTPC is needed for proper formation of apicoplast-ER contact sites and seems to mediate the transference of Ca 2+ between these organelles [57]. Parasites lacking TgTPC lose their apicoplast and have a severe growth defect that resembles the phenotype of other apicoplast-defective mutants [255], suggesting that TgTPC is necessary to maintain the integrity of the organelle and the fitness of the parasite.
The participation of intracellular channels in organellar crosstalk is emerging as a new physiological role of these proteins in protozoan parasites. The localization, properties, and regulation of these channels is proving to be as unique as the biology of the parasites themselves, and further studies are required to explore the potential of these proteins as selective drug targets.

Master manipulators: Ion channels at the interface with the host cell
A hallmark of parasitic lifestyle is the ability to manipulate the host for the benefit of the parasite and protozoans have developed multiple strategies to ensure their supply of "goodies." The interaction between parasites and their mammalian hosts causes extensive changes to the host cells, mediated by secretion of extracellular vesicles, immunomodulatory effects, changes in gene expression, and modification of cell permeability. The most extensively studied example is the remodeling of erythrocytes during Plasmodium infection, which involves the formation of new permeation pathways (NPPs) [256]. Upon RBC invasion, the parasites establish a parasitophorous vacuole (PV) where they reside for their intraerythrocytic development. Early on during PV formation, parasite-secreted proteins form a pore known as Plasmodium translocon of exported proteins (PTEX) [257][258][259]. This complex facilitates the export of proteins that completely change the physiology of the RBC. This "exportome," consisting of more than 300 proteins and can be divided into four major groups: heat-shock proteins, proteins involved in lipid homeostasis, essential kinases, and proteins that mediate nutrient acquisition (reviewed in [260]). The result is a modification of the RBC intracellular concentrations of Na + and K + [261] with increased permeability to organic cations [262], organic osmolytes [263], and anions [264]. Most of these NPPs are due to the plasmodial surface anion channel (PSAC), a multimeric complex produced by the parasites and exported to the RBC membrane [265]. PSAC contains the pore-forming subunit CLAG3 and two associated proteins RhopH2 and RhopH3 [266], required for trafficking to the cell surface [267,268]. Two main purposes have been postulated for PSAC; one is nutrient uptake of essential molecules required for intracellular growth [264,265,269,270], but the marginal fitness defect caused by CLAG3 knockout in vitro suggests additional roles [271]. Upon insertion of PSAC in the RBC membrane, intracellular Na + concentration increases 10 times, while K + decreases significantly. It is possible that the ionic environment modification is what fuels nutrient uptake and membrane potential regulation by sodium-dependent secondary transporters, located at the parasite cell membrane [62, 272,273]. Electrophysiological analysis of PSAC revealed a voltage sensitive anionic channel with unusually small conductance and with very low permeability to Na + [264,274]. Paradoxically, it has a broad selectivity for sugars, amino acids, purines, and vitamins [269,[275][276][277]. The identity of the pore-forming proteins involved in the PSAC activity is still matter of debate [24,25,278]. Multiple studies addressing the composition and characteristics of the NPPs have given origin to two alternative models. The first one postulates CLAG3 as the poreforming unit responsible for PSAC conductance [264,265,267,270], while the second favors the idea that CLAG3 interacts with chloride channels from the host cell, modulating the activity of the NPPs [279][280][281][282].
Whether PSAC functions to mediate uptake directly or indirectly, what is certain is that, once in the RBC cytosol, nutrients must cross the PV to reach the parasites. In erythrocytic and liver stages, only one protein has been identified as a nutrient pore located in the PV, the exported protein 2 or EXP2. EXP2 is a size-selective channel that allows small molecules up to 1.4 kDa to traverse the PVM, constituting the main permeation pathway for amino acids and sugars [283,284]. It has a large conductance of around 300 pS, and several subconductance states [283]. A particular aspect of EXP2's function is its ability to import nutrients when it forms homomeric complexes, or to interact with Hsp101 and PTEX150 to assemble the PTEX protein-exporting pore [283,285]. While EXP2 seems to be sufficient to form a conductive pore, optimal activity at the PVM requires exported protein 1 (EXP1) [286]. Association between EXP1 and EXP2 is necessary for nutrient uptake, but not for the exporting functions of EXP2. Homologues of EXP2 have been found in T. gondii, where GRA17 and GRA23, dense granule proteins secreted by intracellular tachyzoites, form a nutrient-permeable pore with similar characteristics to EXP2 [287]. The elimination of EXP2 or GRA17 resulted in severe reduction of parasite growth, indicating their essentiality for intracellular proliferation [283,[287][288][289]. Thus, PV forming apicomplexans seem to have developed a common strategy for scavenging resources from their host cell, by establishing large nonselective pores in the membrane of the PV.
In trypanosomatids with intracellular developmental stages, the PV provides a protective environment for Leishmania parasites replicating inside macrophages [290,291], while in T. cruzi, the PV is a transient structure [292]. Leishmania promastigotes infect phagocytic cells by promoting their own endocytosis. After incorporation into the phagolysosome, promastigotes differentiate to amastigotes that replicate intracellularly and live in their protective PV [293]. The maturation of the PV involves an active exchange with the endolysosomal system (reviewed in [39]). Fusion of vesicles with the PV provide an environment rich in sugars, amino acids, and lipids required for amastigote replication [294][295][296][297]. Thus, active nutrient scavenging is restricted to arginine and iron [298], which seem to be accumulated in the PV by the mammalian arginine transporter SLC38A9 and Nramp1, a putative iron pump [298]. The evidence suggest that Leishmania has established a different strategy for nutrient acquisition, promoting the insertion of host cells transport proteins into the PVM, instead of producing and secreting its own pore-forming proteins, as it is the case in apicomplexans.
T. cruzi trypomastigotes invade cells either by a lysosomal membrane repair-induced mechanism [299] or by interaction with surface molecules and activation of PIP 3 -dependent signaling pathways [300]. In both cases, the parasite establishes a PV within the first two hours postinvasion and differentiates into intracellular amastigotes. Shortly after, there is a marked increase in the pH of the acidic vacuole followed by fragmentation of the PV and escape of the amastigotes to the cytosol, where they replicate with full access to host cell nutrients [301]. The disassembly of the PV is presumably triggered by the secretion TcTOX, a protein that has pore-forming activity at acidic pH but whose molecular identity has not been elucidated yet [301].
Each of the strategies summarized above represent a unique form of parasite interaction with their host cells that has evolved to provide a suitable environment for intracellular development. The disruption of these mechanisms for nutrient acquisition affects the growth of the parasites, deserving further attention as they could also be an obvious molecular target for the development of new therapeutic alternatives.

Future perspectives: Ion channels as potential drug targets
Infections caused by protozoan parasites are still a major public health problem, mostly in developing countries where coinfections with other microorganisms are common. Considering the high prevalence of T. gondii (around 30%) and Plasmodium infections averaging over 200 million a year, the combined burden of diseases caused by protozoans affects almost half of the world population. The therapeutic options for treating infections caused by these parasites are limited and have complex administration regimes that rely on drug combinations and long treatments. Finding affordable, selective, and effective oral compounds that do not elicit resistance is the goal of current drug development efforts.
Analysis of therapeutic drugs currently in clinical use show that ion channels represent the second most frequent target after GPCR [302] and have been postulated as suitable drug targets against bacterial, viral, and parasitic infections [303][304][305][306]. L-type Ca 2+ channel blockers verapamil, amlodipine, diltiazem, and nifedipine are the most prescribed treatment for cardiovascular diseases such as hypertension and angina [307,308]. Ion channel modulators are widely used to treat neurological and psychiatric disorders such as epilepsy, neuropathic pain, anxiety, and depression [309][310][311][312][313][314]. They have proven efficacious against helminths, with praziquantel activating TRP channels in Schistosoma [315], ivermectin targeting glutamate-activated chloride channels in a variety of nematodes [316,317], and imidazothiazoles and tetrahydropyrimidines acting as cholinomimetics [318,319]. While currently used antiprotozoal drugs do not target ion channels directly, artemisinin seems to inhibit PfATP6, the P. falciparum homolog of the SERCA pump [320], although some differences between in vitro and in vivo studies question this putative mechanism of action [321,322]. Atovaquone, another antimalarial often used in combination with proguanil, targets the ETC and collapses the mitochondrial membrane potential [323,324]. In trypanosomatids, Ca 2+ channel blockers nifedipine, amlodipine, and amiodarone have shown antiparasitic activity [105,110,112], and Ca 2+ homeostasis has been proposed as a cellular target for drug development [55, 325,326]. Over the years, a number of channels and transporters essential for parasite survival have been identified (Fig 3). The disruption of mechanisms that facilitate nutrient and ion exchange in protozoans is a promising area for therapeutic advances. Targeting molecules localized at the plasma membrane of the host cells, the PV, or the parasites' membranes can interrupt essential processes that sustain viability, growth, and infectivity. The obligated question is, Why aren't we there yet? The study of ion channels and transporters in protozoans has faced important technical limitations derived from the difficulty of applying electrophysiological methods of patch clamp recording to small, often motile, cells. Additionally, while these powerful approaches have single molecule resolution, they are time consuming and require significant expertise [18], so they have been sidelined by high-throughput phenotypic screenings fueled by newly developed whole-genome CRISPR-based techniques. Undoubtedly, these studies have provided valuable information regarding essential genes and pathways in the parasites, (2) those expressed at the plasma membrane of the parasites; and (3) channels found in intracellular organelles. (A) Plasmodium parasites residing intracellularly export proteins like PSAC to the host cell. Thus, parasites control the import of nutrients while also exploiting proteins from the host cell to create favorable conditions for their growth. AQP9 and PIEZO1 activity increases in infected RBC, and VRAC activity is stimulated in liver cells by Plasmodium infection. Blockage of these proteins severely impacts parasite growth, suggesting their potential as drug targets. Similarly, interference with the activity of pore-forming proteins in the PVM such as EXP2 in Plasmodium and GRA17/GRA23 in Toxoplasma gondii blocks nutrient import and parasite growth. (B) In apicomplexans, blockers of the Na + pump ATP4 impact parasite growth and PfATP4 has been shown susceptibility to a large number of compounds. Plasma membrane calcium pump (PMCA) and TPPL2 Ca 2+ channels are essential for T. gondii tachyzoite growth, and so it is the SERCA pump in the ER, showing that Ca 2+ homeostasis is a promising target for drug development. Additional proteins worth to explore in T. gondii are the apicoplast two-pore channel TPC and mitochondrial porin VDAC. (C) In trypanosomatids, Ca 2+ controlling proteins including calcium channels (VGCC, IP 3 receptors, VDAC, and MCU) and the PMCA have been proposed as druggable targets. Expression of H + -ATPases and K + channels with very low homology with mammalian proteins could offer an opportunity for the development of selective inhibitors. Modulation of AQP activity is associated with resistance to anti-leishmania therapy and could be exploited as a permeation pathway for chemotherapy.

PLOS PATHOGENS
but discrepancies between large-scale screenings and targeted functional studies argue for an integrative approach to correlate genetic and physiological data [24]. To speed up the evaluation of ion channels as suitable drug targets in protozoans, systematic studies addressing their structural and functional properties are paramount. Structural similarity rather than homology-based screenings, combined with high-throughput electrophysiology in heterologous and native membranes, would increase our capacity for the discovery of new ion channels and transporters that have evaded identification in protozoans [327,328]. These methods, combined with high resolution-low output tools such as cryo-electron microscopy and singlechannel electrophysiology, would allow us to evaluate channels in situ, in a physiological context, and to pinpoint key differences that can be exploited to increase the selectively and potency of new compounds.
Finally, we must increase our commitment to translational applications of basic biology findings and incorporate new technologies to combat old foes. Repurposing of approved drugs, together with evaluation of new natural and synthetic compounds, offers a financially viable avenue for novel treatments against neglected parasitic diseases. The rapid growth of biologics as therapeutics, including antibodies to treat inflammatory, tumoral, and viral diseases (see complete list here: www.antibodysociety.org/antibody-therapeutics-product-data), brings a new tool that should be also considered as an alternative against parasitic diseases. For example, nanobodies targeting P2X channels have shown promising results against some types of cancer and inflammatory diseases [329][330][331][332].
Infectious diseases are still the most prevalent diseases in the world, and their treatment presents unique challenges driven by our limited understanding of host-pathogens dynamics, the influence of the host microbiomes, and the emergence of resistance, among other biological and socioeconomic factors. What we learn about the role of ion channels in protozoans and their potential as therapeutic targets can offer insights into new treatments for bacterial and fungal infections.